The hybridization of complementary strands of DNA is the underlying principle of all microarray-based techniques for the analysis of DNA variation. In this paper, we study how probe immobilization at surfaces, specifically probe density, influences the kinetics of target capture using surface plasmon resonance (SPR) spectroscopy, an in situ label-free optical method. Probe density is controlled by varying immobilization conditions, including solution ionic strength, interfacial electrostatic potential and whether duplex or single stranded oligonucleotides are used. Independent of which probe immobilization strategy is used, we find that DNA films of equal probe density exhibit reproducible efficiencies and reproducible kinetics for probe/target hybridization. However, hybridization depends strongly on probe density in both the efficiency of duplex formation and the kinetics of target capture. We propose that probe density effects may account for the observed variation in target-capture rates, which have previously been attributed to thermodynamic effects.

We investigate how probe density influences hybridization for unlabeled target oligonucleotides that contain mismatched sequences or targets that access different binding locations on the immobilized probe. We find strong probe density effects influencing not only the efficiency of hybridization but also the kinetics of capture. Probe surfaces are used repeatedly, and the potentially large contributions of sample-to-sample variations in surface heterogeneity and nonspecific adsorption are addressed. Results of kinetic, equilibrium, and temperature-dependent studies, obtained using in-situ surface plasmon resonance (SPR) spectroscopy, show that hybridization for surface immobilized DNA is quite different from the well-studied solution-phase reaction. Surface hybridization depends strongly on the target sequence and probe density. Much of the data can be explained by the presence of steric crowding at high probe density; however, the behavior of mismatched sequences cannot be understood using standard models of hybridization even at the lowest density studied. In addition to unusual capture kinetics observed for the mismatched targets, we find that the binding isotherms can be fit only if a heterogeneous model is used. For mismatched targets, the Sips model adequately describes probe-target binding isotherms; for perfectly matched targets, the Langmuir model can be used.

We report a quantitative study of the kinetics of formation for a two-component tethered ssDNA monolayer film using in situ two-color surface plasmon resonance (SPR) spectroscopy. The attachment of the DNA to gold is facilitated by functionalization at the 5′ end with a thiol group connected by a hexamethylene linker (HS-C6-ssDNA). Detailed data analysis is performed by quantitative comparison of the DNA coverage versus time kinetic data obtained from SPRS with numerical solutions for the differential equations for simultaneous adsorption, desorption, and diffusion at the interface. The kinetics of adsorption of HS-C 6 -ssDNA onto bare gold as well as the kinetics of loss of HS-C 6 -ssDNA from the surface during subsequent treatment with mercaptohexanol can be understood in terms of a simple physical model and self-consistent parameters. The kinetics of HS-C 6 -ssDNA adsorption on bare gold are compared to the kinetics of hybridization of surfaceattached thiolated ssDNA with the fully complementary ssDNA in free solution and found to follow remarkably similar kinetic pathways. In contrast, the adsorption of ssDNA follows complex kinetics that cannot be modeled with a single kinetic step. That is, the presence of a thiol functionality on a 25-mer ssDNA gives rise to adsorption behavior that is clearly kinetically distinct from simple ssDNA adsorption on gold.

We demonstrate that in situ optical surface plasmon resonance spectroscopy can be used to monitor hybridization kinetics for unlabeled DNA in tethered monolayer nucleic acid films on gold in the presence of an applied electrostatic field. The dc field can enhance or retard hybridization and can also denature surfaceimmobilized DNA duplexes. Discrimination between matched and mismatched hybrids is achieved by simple adjustment of the electrode potential. Although the electric field at the interface is extremely large, the tethered single-stranded DNA thiol probes remain bound and can be reused for subsequent hybridization reactions without loss of efficiency. Only capacitive charging currents are drawn; redox reactions are avoided by maintaining the gold electrode potential within the ideally polarizable region. Because of potential-induced changes in the shape of the surface plasmon resonance curve, we account for the full curve rather than simply the shift in the resonance minimum.electrochemistry ͉ DNA hybridization ͉ DNA mismatch discrimination I ncreasing research efforts are directed toward the detection of nucleic acid interactions with immobilized oligonucleotide probes for DNA microarray applications (1, 2). Limitations of most current technologies include complex DNA immobilization procedures, the need for fluorescence or other labeling, and slow hybridization kinetics that require long incubation times. In addition, those experimental conditions that optimize DNA duplex formation often also reduce the stringency of hybridization. That is, for oligonucleotides that are sufficiently long to distinguish a particular sequence in the presence of unrelated DNA, a mismatched base pair has only a marginal effect on the stability of the duplex (3). In DNA microarray applications, mismatched hybrids lead to ''false positives.''The use of an electric field to easily control the electrostatic forces on surface-immobilized polyelectrolytes such as DNA has not yet been fully exploited as a means for improving the speed and stringency of biomolecular interactions at interfaces.In this paper, we investigate the effect of simple electrostatic charging on the interactions of surface-bound monolayer nucleic acid films with unlabeled DNA target oligonucleotides by using a combination of electrochemical control and in situ real-time surface plasmon resonance (SPR) spectroscopy detection.The monolayer DNA thiol films used in this work are tethered directly to the SPR metal sensor surface through a gold-thiol covalent attachment. Therefore, the immobilized DNA hybrids are exposed to a field gradient at the metal͞electrolyte interface on the order of 10 9 V͞m. We show that this field can be used, in a reversible manner, to increase or decrease the rate of oligonucleotide hybridization. In addition, we show that a repulsive potential preferentially denatures mismatched DNA hybrids within a few minutes, while leaving the fully complementary hybrids largely intact. This sequence selectivity in hybrid denaturation imparts an extr...

No abstract

Background: A critical challenge in cell biology is quantifying the interactions of cells with their extracellular matrix (ECM) environment and the active remodeling by cells of their ECM. Fluorescence microscopy is a commonly employed technique for examining cell-matrix interactions. A label-free imaging method would provide an alternative that would eliminate the requirement of transfected cells and modified biological molecules, and if collected nondestructively, would allow long term observation and analysis of live cells.

BackgroundSurface plasmon resonance imaging (SPRI) is a label-free technique that can image refractive index changes at an interface. We have previously used SPRI to study the dynamics of cell-substratum interactions. However, characterization of spatial resolution in 3 dimensions is necessary to quantitatively interpret SPR images. Spatial resolution is complicated by the asymmetric propagation length of surface plasmons in the x and y dimensions leading to image degradation in one direction. Inferring the distance of intracellular organelles and other subcellular features from the interface by SPRI is complicated by uncertainties regarding the detection of the evanescent wave decay into cells. This study provides an experimental basis for characterizing the resolution of an SPR imaging system in the lateral and distal dimensions and demonstrates a novel approach for resolving sub-micrometer cellular structures by SPRI. The SPRI resolution here is distinct in its ability to visualize subcellular structures that are in proximity to a surface, which is comparable with that of total internal reflection fluorescence (TIRF) microscopy but has the advantage of no fluorescent labels.ResultsAn SPR imaging system was designed that uses a high numerical aperture objective lens to image cells and a digital light projector to pattern the angle of the incident excitation on the sample. Cellular components such as focal adhesions, nucleus, and cellular secretions are visualized. The point spread function of polymeric nanoparticle beads indicates near-diffraction limited spatial resolution. To characterize the z-axis response, we used micrometer scale polymeric beads with a refractive index similar to cells as reference materials to determine the detection limit of the SPR field as a function of distance from the substrate. Multi-wavelength measurements of these microspheres show that it is possible to tailor the effective depth of penetration of the evanescent wave into the cellular environment.ConclusionWe describe how the use of patterned incident light provides SPRI at high spatial resolution, and we characterize a finite limit of detection for penetration depth. We demonstrate the application of a novel technique that allows unprecedented subcellular detail for SPRI, and enables a quantitative interpretation of SPRI for subcellular imaging.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2121-15-35) contains supplementary material, which is available to authorized users.

Glycoanalysis is important in the manufacture and quality control of protein therapeutics. An emerging method for glycoanalysis is the use of lectin arrays. Critical to the performance of these arrays is the immobilization of lectin molecules. Polydopamine has recently been shown to adsorb to a wide variety of surfaces. In this study, polydopamine (pDA) was used to modify gold, indium, and iridium surfaces and promote the adhesion of the alpha-mannose-specific lectin concanavalin A (Con A). The activity of the surface-bound lectin was demonstrated with the alpha-mannose-presenting glycoprotein ribonuclease B (RNase B). Surface plasmon resonance spectroscopy (SPRS) was used to demonstrate the selective affinity of RNase B for Con A. Surface-MALDI-TOF MS experiments revealed that the affinity of polydopamine-immobilized Con A for the glycoforms of RNase B is significantly affected by slight variations in oligosaccharide structure and composition. Specifically, surface-bound Con A binds certain Man7, Man8, and Man9 RNase B glycoforms more strongly than Man5 and Man6 glycoforms.